The increasingly widespread use of advanced spectroscopic detectors such as mass spectrometers has dramatically broadened the utility and information-yield of analytical liquid chromatographic separations. Older “conventional” detection techniques such as refractometry or fixed-wavelength ultraviolet-absorbance detection might imply the presence of a suitable analyte within the detection volume, and with the use of known calibrants, might imply the concentration of that analyte. However, the identity of the analyte, at best, was inferred by comparison with the chromatographic retention time or retention volume of a known standard. Identification of the analyte was effectively not a property or a capability of those older detection subsystems.
Such detectors were also susceptible to significant quantitation errors in the presence of overlapping chromatographic bands or zones, limiting their utility in the analysis of highly-complex mixtures as are commonly encountered in biological and environmental applications. Historically, the application of mass spectrometry to the task of liquid chromatography detection facilitated analyte detection within vastly more complex mixtures, by permitting high-resolution separation in the mass-to-charge domain to augment chromatographic separation in the liquid-volume domain.
Depending upon the mode of mass spectrometric analysis being performed, and the nature of the analyte(s), putative compound identification might be performed in-line during the separation, substantially without reliance upon the chromatographic retention time or retention volume of a standard. There exist, however, important classes of compounds for which mass spectrometry, alone, is not capable of rendering a full and complete identification. Commonly, examples are found where isomerization is present, where the isomers share or exhibit the same chemical formula and parent mass-to-charge-ratio, but are assembled in arrangements which may be either structurally or spatially distinct. Knowledge of the chemical formula, while useful, is incomplete if the configuration or arrangement of the molecule is questionable or fully unknown. Isomers may have distinctly (in some cases, radically) different behaviors within biological systems, if supplied as pharmaceutical compounds, or if rendered as degradation products. The fates of important contaminant materials in the environment may entail molecular rearrangements of multiple types.
Nuclear Magnetic Resonance (“NMR”) spectroscopy is a powerful complement to mass spectroscopy in the analytical toolkit. The structural information yielded by NMR spectra can be used to infer how the molecule is arranged. However, the requirements for NMR analysis have made it difficult to directly couple NMR analysis with other detectors and chromatographic techniques. In this context, the term “directly coupled” means a single sample is separated by chromatographic means to form a separated sample and one or more aliquots of the separated sample are received by spectrometric and NMR analysis without an intervening collection into containers such as vials, well-plates, and the like. The use of vials, well-plates, or other fraction-collection devices implies an “off-line” analytical methodology, in contrast to a directly-coupled methodology. In a directly-coupled method, the analyte sample borne in solution in a mobile phase is conveyed within fluid conduits throughout that analytical method, and typically does not emerge into a receiving vessel such as a vial until it has traversed the entirety of that method. A vial may be used to collect waste from a first analytical process or method, or a vial may be used to capture the sample for transport and submission to a subsequent analytical method implemented as a separate system. The subsequent system would be referred to as operating “off-line” from the first system.